Immune surveillance for six vaccinable pathogens using paired plasma and dried blood spots in HIV infected and uninfected children in Kinshasa

Child vaccination reduces infant mortality rates. HIV-infected children present higher risk of diseases than non-infected. We report the protection coverage rates for 6 vaccine-preventable diseases in a paediatric population from the Democratic Republic of the Congo (DRC) and the impact of HIV infection, providing the first data on the validity of dried blood samples (DBS) to monitor the immune protection. During 2016–2018 DBS from 143 children/adolescents were collected in Kinshasa (DRC), being 52 HIV-infected. Forty-two had a paired plasma sample. Protective IgG was quantified (VirClia-IgG,VIRCELL) to obtain the optimal cut-off in IgG detection in DBS. ROC curves were generated with R software and statistical analyses with Stata. Protective IgG levels varied across pathogens, not reaching herd immunity. HIV-infected presented lower vaccine protection than uninfected for all analyzed pathogens, except rubella, with statistically significant differences for measles (30.8% vs. 53.8%; p = 0.008) and tetanus (3.8% vs. 22%; p = 0.0034). New cut-offs were calculated when using DBS to improve test performance. We reinforce the necessity to increase pediatric vaccination coverage in Kinshasa, especially in HIV seropositive, with less capacity to maintain adequate antibody levels. DBS were useful to monitor vaccination coverage in seroprevalence studies in resource-limited settings, after optimizing the cut-off value for each pathogen.

vaccine coverage. The DRC vaccination schedule does not yet contemplate the second dose of MCV 9 , used to assess the ability to continue immunization services between the second and fifth year of life 3 .
Children living with the human immunodeficiency virus (HIV) tend to have lesser vaccine protection against vaccine-preventable diseases vs. unexposed children 10 . Thus, it is important to optimize the vaccination schedule, especially in countries with a high rate of paediatric HIV infections. Protective titers after vaccine immunization in HIV-infected children varies depending on the vaccine, immune recovery and viral suppression 11 . Revaccination after initiating antiretroviral therapy (ART) could help to increase protection in HIV-infected children 12,13 . Most of them live in low-middle income countries, where scarce laboratory resources and limited conditions for collecting and handling serum/plasma from blood, which makes difficult to monitor the real paediatric vaccination coverage. In these circumstances, dried blood samples (DBS) could represent a convenient sample for antibody detection 14 , being easy to collect, store and transport without cold chain 15 . DBS have been used in the surveillance and monitoring of numerous infectious diseases [14][15][16][17][18][19][20] . The good correlation between antibody concentrations in DBS and serum/plasma samples support the wider use of DBS in post-vaccination and seroepidemiological studies 14 .
Our study reports the immune seroprotection rates to 6 vaccine-preventable diseases (diphtheria, tetanus, pertussis, measles, mumps, rubella) in a paediatric and adolescent population in Kinshasa (DRC), analysing the impact of HIV infection. Furthermore, we provide the first data on the validity of DBS to test the immune protection to these 6 pathogens, establishing the cut-off values that provide optimal sensitivity and/or specificity in DBS for each infection in the study cohort.

Clinical and laboratory procedures. DBS from 143 children and adolescents under clinical follow-up in
Monkole and Kalembelembe hospitals (Kinshasa, DRC) were collected between 2016 and 2018, as previously described 21 . Forty-two of them (29.4%) also had paired plasma. All specimens had associated clinical-epidemiological data. The samples were stored in Kinshasa hospitals at − 20 °C until their transport to Madrid, where they were kept at − 80 °C until processing to analyse and quantify the presence of protective IgG against six pathogens responsible for vaccine-preventable diseases: diphtheria, tetanus, pertussis, measles, mumps and rubella.
The first HIV diagnosis was performed in Kinshasa by three rapid serological tests: Determine™HIV-1/2Ag/ Ab (ALERE), Double-Check Gold HIV1&2 (ORGENICS) and Uni-Gold HIV (TRINITY BIOTECH) in patients older than 18 months of age. In infants under 18 months, the 4th generation immunoassay VIDAS® HIV Duo Ultra (BIOMERIEUX) or, exceptionally, the molecular test ABBOTT real-time HIV-1 Qualitative were used.
In the HIV-1 Molecular Epidemiology Laboratory (Madrid), the serological status of the children was confirmed by the Geenius™HIV-1/2 (BIO-RAD) assay. One whole blood spot was taken from each card and eluted in 150 µl NucliSens easyMAG elution buffer 3 (BIOMERIEUX) for 1 h with rotation. The test was performed according to manufacturer indications, but using 40 µl of the eluted, as previously published by our group 19 . The results obtained by Geenius™HIV-1/2 were confirmed in all seropositive and indeterminate patients using the molecular point-of-care XpertQual (CEPHEID), which provides a binary "detected"/"not detected", according to manufacturer's instructions.
Quantification of IgG against six vaccinable diseases. VirClia-IgG (VIRCELL) technique, an indirect chemiluminescent immunoassay to detect the antibodies present in the sample with the antigen bound to the polystyrene surface, was used to quantify the presence of protective IgG against the six pathogens with 5 µl of plasma. When using DBS, one dot (70 µl of whole blood containing 42.7 µl plasma after considering 39% haematocrit 22 ) was eluted in 770 µl of phosphate-buffered saline (PBS), being tested 100 µl of the final elution in each specific VirClia-IgG test (equivalent to 5 µl plasma per test). We considered as gold standard the cut-off values provided by VirClia for the six pathogens in plasma/serum (Table 1), which provides 96-100% sensitivity and 100% specificity. We considered that a child had protective IgG levels for a pathogen under study, or concentration of antibody titers with sufficient affinity required to neutralize the pathogen 8 , when the anti-pathogen IgG levels in his plasma were included in the range of positivity proposed by VIRCELL and specified in the technical file of each specific VirClia-IgG assay.
To optimize the DBS use for IgG measurements, the sensitivity, specificity and positive (PPV) and negative (NPV) predictive value of the tests were calculated for each of the six measurements in DBS of the 42 children and adolescent under study with paired plasma/DBS, considering as reference the results obtained in plasma Immunization level in plasma of the paediatric population. Protective IgG levels greatly varied for each pathogen in the study cohort. Different protection coverage rates, or % of subjects with immune response against an infection and/or disease 23 , was found in the 42 children and adolescents with available plasma for rubella (93%), diphtheria (71%), mumps (69%), measles (64%), tetanus (7%) and pertussis (2%) in plasma (Fig. 1A). Therefore, we found that 93% of children/adolescents did not show IgG protection against pertussis, 43% against tetanus, 29% against measles and 21% against mumps. The percentage of patients with indeterminate results in plasma was variable, ranging from 2% for rubella to 50% for tetanus (Fig. 1A). Indeterminate values were excluded from the final calculation.
Cut-off calculation for each pathogen using DBS. To evaluate the validity of DBS to monitor the immune protection, we also analyzed the presence of protective IgG against the six pathogens in DBS from the 42 subjects with available paired DBS/plasma specimens (Fig. 1B). The sensitivity of each VirClia-IgG test for www.nature.com/scientificreports/ the detection of antibodies in DBS, considering the same cut-off for plasma (gold-standard sample) established by the manufacture's, was 100% for pertussis, diphtheria and mumps, 97.4% for rubella and lower for measles (81.8%) and tetanus (66.7%). Specificity was very high for pertussis (97.6%), but decreased for tetanus (87.2%), measles (40%), mumps (7.7%), diphtheria and rubella (0% each). The PPV ranged from 28.6% (tetanus) to 92.7% (rubella) ( Table 3).
A new cut-off (optimal cut-off) was calculated for each test to achieve optimal sensitivity and/or specificity in the detection of IgG using DBS. The application of optimal cut-off for each pathogen allowed to increase the PPV and NPV of the tests from the study cohort when using DBS ( Table 3).
Comparison of immunization results in DBS vs. plasma in the 42 children and adolescents with paired samples. The agreement in the immune protection rate for the 6 pathogens under study in the 42 children with paired DBS/plasma specimens varied according to the considered cut-off value showed in Table 3. Supplementary Table 2 provides the specific correlation between plasma and DBS in each child with paired DBS/ plasma, highlighting these HIV positive children and considering different cut-offs: VIRCELL (see Table 1), optimum (see Table 3), and maximum specificity (see Table 3  www.nature.com/scientificreports/ When using the plasma cut-off for DBS, the percentage of immunized children in DBS for all pathogens was higher than when using plasma ( Fig. 2 options A vs. B), with significant differences for 2 of them (+ 28.6% for mumps and diphtheria). By contrast, the observed protection was only + 2.4% for pertussis, + 4.8% for rubella and + 9.5% for tetanus and measles.
When the optimal cut-off showed in Table 3 for each pathogen was applied in DBS ( Fig. 2 option D), the percentage of immunized among these 42 children was lower than using plasma to diphtheria (− 35.7%), mumps and measles (− 23.8%) and rubella (− 4.8%), was slightly higher (+ 2.4%) to tetanus, and similar to pertussis.

Estimated immunization level in DBS of 143 children and adolescents.
To study the protection coverage rates to 6 vaccine-preventable diseases in the whole study population from Kinshasa, we evaluated the percentage of children with protective IgG against each pathogen under study in the 143 DBS using cut-off providing maximum (100%) specificity (corresponding to 0% false positives) described at Table 3. We decided to use this cut-off to guarantee that all patients identified as immunized in the whole study cohort were protected, avoiding overestimation in the percentage of immunized patients. We did not use the plasma cut-off due to the previously observed overestimation of the percentage of immunized for all pathogens (Fig. 2 option B). Supplementary Table 3 provides the specific correlation between levels of reactive IgG in DBS from 143 children according to their HIV status and considering both maximum specificity and optimal cut-offs provided in Table 3.
With this 100% specificity cut-off, protective IgG were detected in less than half of the 143 studied patients: 46.9% for rubella, 45.5% for measles, 36.4% for diphtheria, 24.5% mumps, and 0.7% for pertussis. As it happened with the immunization values obtained when comparing the 42 paired samples DBS vs. plasma, immunization rates were between 2 and 3 times lower than those observed in plasma (gold standard) comparing data from the 143 DBS vs. 42 plasma (15.4% vs.7.1%), except for tetanus, where the percentage of immunized patients doubled (Fig. 3).

Impact of HIV infection in protection rate to 6 vaccine-preventable pathogens.
Based on the analysis in the 143 DBS using the cut-off providing maximum (100%) specificity (Fig. 4A), we explored the impact of HIV infection in the protection coverage rates to the 6 vaccine-preventable diseases. Protective IgG to each pathogen were measured in 52 HIV-infected vs. 91 uninfected children of the study population from Kinshasa. We observed lower vaccine protection in HIV infected children except for rubella, which was slightly higher. The differences were statistically significant for measles (30.8% vs. 53.8%, p = 0.008) and for tetanus (3.8% Table 3. Results of VirClia-IgG test for the detection of protective IgG against six pathogens responsible for vaccine-preventable diseases using dried blood samples from 42 paediatric patients. IU International units, ml Millilitre, PPV Positive predictive value, NPV Negative predictive value, DBS Dried blood spots sample, Max Maximum, Sen Sensitivity, Spe Specificity. The cut-offs provided by VIRCELL for plasma have been taken as reference (see Table 1). 100% values are in bold. % of paƟents with pathogen immunity Figure 3. Comparison of the percentage of patients with pathogen immunity in the study population (42 plasmas vs. 143 DBS) using the optimal and maximum specificity cut-offs. The plasma cut-off provided by VIRCELL for each pathogen is shown in Table 1. The optimal or maximum specificity cut-offs calculated for each pathogen are indicated in Table 3. Statistically significant differences found: *p = 0.01-0.03; **p = 0.001-0.003; ***p < 0.0001. % of paƟents with pathogen immunity % of p p p p p p p p p p p p p paƟ aƟe e e e ents ts s with h h pat pat pat p p pat p p p p p p hog g g g g g g g g g g ge e en immunity Figure 2. Pathogen immunity in 42 children and adolescents from Kinshasa with paired plasma/DBS considering different cut-offs. The plasma cut-off provided by VIRCELL for each pathogen is shown in Table 1.

OpƟons S ample
Options A and B, with the VIRCELL cut-off according to Table 1. Options C and D, with the maximum specificity or optimum cut-offs calculated for each pathogen, as indicated in Table 3. Statistically significant differences found: *p = 0.01-0.03; **p = 0.001-0.003; ***p < 0.0001.

Discussion
The evaluation of immunization programs is key to identify areas of suboptimal vaccination coverage 24 . Our pilot study provides pioneer data related to the seroprotection to six infectious diseases preventable by vaccination in a cohort of children and adolescents living in Kinshasa (DRC) using samples collected during 2016-2018. The quantification of IgG levels in DBS did not present any difficulty compared to plasma, and we detected IgG in the plasma fraction present in the dried blood drop collected on Whatman paper, as we previously did for other pathogens 20,21,[25][26][27] .
Our results reveal that IgG levels greatly varied for each pathogen in the study population, not reaching the percentage necessary to achieve herd immunity, considering this as the minimum number of IgG reactive individuals necessary to avoid the pathogen spreading in the population. We also observed lower vaccine protection in HIV infected children vs. uninfected. We confirmed the usefulness of DBS in the absence of plasma/serum to study seroprevalence against vaccinable-preventive diseases. The DBS use for seroprevalence surveillance studies with this approach, and after optimizing the cut-off value for each pathogen, would allow for targeted vaccination campaigns, which could potentially provide services to the communities most in need and reduce vaccine campaign cost. We also provided the guideline for the adaptation of VIRCELL assays from serum to DBS, and the optimal pre-analytical treatment of DBS specimens for clinical vaccine or seroepidemiological studies. www.nature.com/scientificreports/ Control of airborne viral infections with a very high reproduction number, like measles, mumps or rubella, requires high vaccine coverage 28,29 . Herd immunity, or the minimum number of individuals with pathogen immunity necessary to avoid the pathogen spreading in the population, has been previously defined [30][31][32][33][34] . Herd immunity threshold differs across pathogens: 91-94% for measles, 86-93% for mumps, 83-94% for rubella, 75-80% for diphtheria, and 90-94% for pertussis 32 . However, herd immunity thresholds and reproduction number can vary slightly across authors for some pathogens 32,33 . Clostridium tetani is not communicable between human hosts and there is no threshold proportion of immune persons below 100% that can ensure total absence of tetanus from a community 33 Despite measles is endemic in the DRC, and 91-94% herd immunity is required to halt its transmission, coverage not reached in many African countries 35 . Among the 42 children and adolescents from Kinshasa with available plasma, 7 out of 10 were immunized against mumps and 9 out of 10 against rubella (Fig. 2), despite the absence of both vaccines in the DRC vaccination schedule 36 , revealing that protection was due to pathogen exposition and not by vaccination. We found that 6 out of 10 were immunized against measles, whose vaccine is administered at 9 months of age in the RDC.
Antibodies against measles, mumps and rubella can be present for up to 20 years after trivalent vaccine administration 37 , decreasing in some children when the vaccines are given separately 38 . Although MMR vaccineinduced protection appears to persist at least into early adulthood, constant monitoring of protection against these pathogens is necessary 37 . In addition, it must be taken into account that the current vaccination schedule during the first year of life of older patients under study could differ from the current one given to younger participants, modifying the protection against some of the vaccine pathogens. Our data revealed similar immunization rates to measles (64%) in the study paediatric population from Kinshasa during 2016-2018 than in other paediatric cohorts in the DRC during 2013-2014 39 , being slightly higher than the vaccination coverage after the first and single dose of measles vaccine administered at 9 months of age reported by the WHO in that country (57%) 9 .
DTP3 coverage in the DRC, used as an indicator of the capacity of a country to provide immunization services, reached 57% in 2019 9 . However, we found different immunization rates for each pathogen in the 42 plasma from the study population, being 71.4% of them immunized against diphtheria, but only 7.1% against tetanus and 2.4% against pertussis. If patients protected against diphtheria had suffered from the disease was not reported in the available clinical files. The high vulnerability to tetanus and pertussis of the Congolese paediatric and adolescent population is consistent with the latest official data from the country 9 .
The interference with other infectious diseases affects the loss of seroprotection [40][41][42][43][44] . In this regard, we wanted to analyse the impact of HIV infection on seroprotection levels against the 6 pathogens in our paediatric cohort from Kinshasa. To avoid the loss of statistical robustness of the results due to the low sample size using plasma samples (only in 42 patients), we used DBS, the available sample in all 143 subjects under study. We considered the cut-off providing 100% specificity to guarantee that all patients identified as immunized were protected and to avoid overestimation in the rate of immunized children and adolescents in the complete study cohort. However, it is important to note that the cut-off providing 100% specificity in the detection of protective IgG for each pathogen in DBS might be always calculated in each study population with paired plasma/DBS samples and only be extrapolated to other population with similar prevalence for each analyzed infection.
The effect of HIV-1 exposure and antiretroviral treatment strategies in HIV-infected children on the immunogenicity of vaccines during infancy has been also reviewed, showing differences in protection 45 . Since HIV infection impairs humoral and cell-mediated immune functions early in infancy 46,47 , the poor antibody levels to different vaccines found in our study could be probably related to major immunological dysfunction involving both cellular and humoral responses in the children under study. Some authors have analyzed the persistence of antibodies to the vaccines in HIV-infected and HIV-exposed uninfected children who previously received these vaccines in routine clinical practice, also reporting lower antibody concentrations after vaccination against some vaccine-preventable diseases in HIV-infected vs. uninfected children 48 . Furthermore, low measles antibody concentration in HIV-infected children has been associated with severe immunodeficiency (< 25% CD4 + T cells, HIV VL ≥ 10,000 copies) in HIV-infected children, as well as to the short duration of antiretroviral treatment 48 . Unfortunately, CD4 data at sampling was not reported in the clinical files of the HIV-infected children under study.
Since children living with HIV present a higher risk of vaccine-preventable infectious diseases than noninfected 10 , vaccination with an adapted schedule, revaccination and periodic monitoring of their seroprotective status are recommended 13,48,49 . The low levels of seroprotection for all pathogens except rubella observed in the 52 HIV-infected patients vs. 91 HIV-uninfected could be explained by the lower capacity of the children with HIV to maintain the titers at adequate levels over time 48 .
Regarding measles, low persistence of antibodies to the measles vaccine has been observed mainly in HIVinfected but also in HIV-exposed children born to HIV-infected mothers 48,50 . The level of measles immunity at birth in both HIV-infected and HIV-1 exposed children would depend on the level of antibodies in the HIVinfected mother and on the extent of placental transfer 50 . In HIV-infected and exposed children, maternal HIV infection might reduce levels of measles antibodies in newborns, and the low levels of measles antibodies at birth would render children susceptible to measles infection at an early age 50 . In HIV-infected children increases the number of CD4 + T cells and B cells during ART, whereas the total immunoglobulin levels decline. Despite immune reconstitution, antibodies against live-attenuated vaccines and wild-type natural virus strains disappear over time in up to 40% of children with HIV-1 infection 51 . Fewer HIV-infected children were protected after vaccination at 12 months than HIV-exposed but uninfected children have been reported, as well as an increase in the proportion of seropositive children increased with increasing age at vaccination among HIV-exposed but uninfected and HIV-unexposed children 52 . Another study showed that the majority of HIV-infected children vaccinated against measles in Morocco develop a suboptimal seroprotective titer 53  www.nature.com/scientificreports/ This study has several limitations. Firstly, plasma was only available in 42 of 143 subjects under study since we used retrospective and remaining samples from other studies in paediatric population in Kinshasa 21,54-56 . Unfortunately, the number of received doses in each enrolled child was not reported in the clinical files, preventing distinguishing the children and adolescents with protective IgG due to vaccination or due to infection. Secondly, the number of positive/negative samples for each pathogen in our study population was very uneven among the 42 with available plasma specimens, ranging from only 1 subject infected with pertussis to 39 infected to rubella. Thus, future similar studies should include a higher number of subjects and a similar number of infected and uninfected children per infection, for a better adjustment of the cut-off values for each pathogen. Thirdly, the cut-off values providing 100% specificity in DBS for each pathogen could not be appropriate for other cohorts with different prevalence of the studied vaccinable-preventive diseases. Cut-off values providing 100% specificity should be always calculated in paired DBS/plasma specimens for the same setting or study cohort with a similar prevalence of vaccinable-preventive diseases under study. In addition, the provided new cut-offs for DBS would be VIRCELL-specific and should not be extrapolated to other similar assays for seroprevalence studies.

Conclusions
Our data reinforce the necessity to increase/improve vaccination coverage in Kinshasa, paying special attention to the HIV-infected paediatric population, with less capacity to maintain antibody titers at adequate levels. To prevent future outbreaks and to avoid preventable deaths from vaccination, we emphasize the urgency for periodic monitoring immunity to vaccine-preventable diseases globally and mainly in countries with limited resources. The use of DBS could help to expand antibody detection for monitoring seroprotection level coverage among children in resource-limited settings when plasma/serum collection is complex or absent. However, the cut-off values should be optimized when DBS are used for seroprevalence studies. Finally, we recommend carrying out new similar studies with a larger sample size with available paired plasma/DBS specimens per subject and vaccination data, and an equitable number of vaccinated and unvaccinated patients to assign a definitive cut-off value for each pathogen in DBS. The procedures described here could be incorporated into existing data collection protocols in the DRC to monitor vaccinable-preventive disease exposures, herd immunity, and effectiveness of immunization programs. www.nature.com/scientificreports/